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ANP32B Deficiency Impairs Proliferation and Suppresses Tumor Progression by Regulating AKT Phosphorylation

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ANP32B Deficiency Impairs Proliferation and Suppresses Tumor Progression by Regulating AKT Phosphorylation Citation: Cell Death and Disease (2016) 7, e2082; doi:10.1038/cddis.2016.8 OPEN & 2016 Macmillan Publishers Limited All rights reserved 2041-4889/16 www.nature.com/cddis ANP32B deficiency impairs proliferation and suppresses tumor progression by regulating AKT phosphorylation S Yang1,6, L Zhou2,6, PT Reilly3, S-M Shen1,PHe1, X-N Zhu1, C-X Li1, L-S Wang4, TW Mak5, G-Q Chen*,1 and Y Yu*,1 The acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) is reported to impact normal development, with Anp32b-knockout mice exhibiting smaller size and premature aging. However, its cellular and molecular mechanisms, especially its potential roles in tumorigenesis, remain largely unclear. Here, we utilize 'knockout' models, RNAi silencing and clinical cohorts to more closely investigate the role of this enigmatic factor in cell proliferation and cancer phenotypes. We report that, compared with Anp32b wild- type (Anp32b+/+) littermates, a broad panel of tissues in Anp32b-deficient (Anp32b− / −) mice are demonstrated hypoplasia. Anp32b− / − mouse embryo fibroblast cell has a slower proliferation, even after oncogenic immortalization. ANP32B knockdown also significantly inhibits in vitro and in vivo growth of cancer cells by inducing G1 arrest. In line with this, ANP32B protein has higher expression in malignant tissues than adjacent normal tissues from a cohort of breast cancer patients, and its expression level positively correlates with their histopathological grades. Moreover, ANP32B deficiency downregulates AKT phosphorylation, which involves its regulating effect on cell growth. Collectively, our findings suggest that ANP32B is an oncogene and a potential therapeutic target for breast cancer treatment. Cell Death and Disease (2016) 7, e2082; doi:10.1038/cddis.2016.8; published online 4 February 2016 The acidic leucine-rich nuclear phosphoprotein 32 kDa lethality and reduced body weight,22–25 indicating a greater (ANP32) protein family are characterized by a N-terminal importance of Anp32b in normal development. In addition, leucine-rich repeat domain and a C-terminal low-complexity gene expression analysis indicates that elevated ANP32B acidic region.1 In mammals, the ANP32 family has at least mRNA expression correlates with highly proliferative three members named ANP32A, ANP32B and ANP32E, tissues.22 We also showed that ANP32B acts as a negative and they regulate a wide spectrum of biological processes regulator of leukemic cell apoptosis,8 and inhibits all-trans – – including chromatin regulation,2 6 caspase activation,7 9 retinoic acid induced leukemic cell differentiation.26,27 protein phosphatase inhibition10–12 and intracellular Although these studies strongly suggested ANP32B as a transport.13,14 Although early investigations suggested that master regulator of cell fate determination, its cellular and three ANP32 members functionally overlap,10 they are molecular mechanisms are still not understood. Considering reported to have diverse roles in cancer progression. ANP32A that some physiological and pathological processes share was shown to inhibit cell transformation15–17 and has reduced many common molecular regulators,28 and ANP32B mRNA expression in prostate and breast cancer.18,19 ANP32E was expression is a marker for aggressive breast cancer,22 we reported to have enhanced expression in gastric cancer,20 and proposed that ANP32B also functions in breast cancer. Here, a high expression of ANP32E was associated with better we used Anp32b-knockout mice, multiple breast cancer cell survival rate in follicular lymphoma.21 Previously we reported lines and clinical patient samples to uncover the potential role that ANP32B, also designated as PHAPI2 or SSP29, is a for ANP32B in cell proliferation of both mouse embryo negative prognostic indicator for human breast cancer.22 Full fibroblasts (MEFs) and breast cancer cells, and find that loss analysis of the expression and functional role of ANP32B in of ANP32B by knockout or RNAi silencing reduced rates of cell cancer progression has still not been undertaken. proliferation. We also show that RNAi silencing induces an Knockout mouse studies demonstrated that loss of Anp32b, extended G1-phase of the cell cycle. In addition, phosphoryla- but not Anp32a and Anp32e, caused a high degree of perinatal tion of AKT, an upstream regulator of cell cycle-associated 1Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai, China; 2Department of Surgery, Branch of Shanghai First People’s Hospital, SJTU-SM, Shanghai, China; 3Laboratory of Inflammation Biology, National Cancer Centre Singapore, Singapore, Singapore; 4State Key Laboratory of Genetic Engineering, Minhang Hospital, Fudan University, Shanghai, China and 5Campbell Family Cancer Research Institute, University Health Network, Toronto, ON, Canada *Corresponding author: G-Q Chen or Y Yu, Department of Pathophysiology, Shanghai Jiaotong University School of Medicine, 280, Chong-qing South Road, Shanghai 200025, China. Tel: +86 21 63846590 776573; Fax: +86 21 64154900; E-mail: [email protected] or [email protected] 6These two authors equally contributed to this work. Abbreviations: ANP32, acidic leucine-rich nuclear phosphoprotein 32; BLI, bioluminescence imaging; CCK-8, cell counting kit-8; CDK, cyclin-dependent kinase; DMBA, 7,12-dimethylbenz(a)anthracene; MEFs, mouse embryo fibroblasts; FBS, fetal bovine serum; GFP, green fluorescence protein; IHC, immunohistochemical staining; IRS, immunoreactive score; PHAPI2, putative HLA-DR-associated protein I-2; PHLPP, PH domain leucine-rich repeat protein phosphatase; PDK1, pyruvate dehydrogenase kinase isozyme 1; PP2A, protein phosphatase 2A; PTEN, phosphatase and tensin homolog; SSP29, silver-stainable protein 29 Received 02.10.15; revised 31.12.16; accepted 04.1.16; Edited by J Chipuk ANP32B deficiency suppresses proliferation and tumorigenesis S Yang et al 2 proteins, is lower coincident with reduced ANP32B upon homozygous Anp32b− / − mice,22 the Balb/c-congenic silencing and in both mouse and human cancers. Anp32b− / − mice also had a statistically significant reduction of body weight compared with wild-type (Anp32b+/+) and +/ − Results heterozygous (Anp32b ) mice at 4 weeks after birth (Figure 1a). Here we also found the decreased weigh of the − − Anp32b− / − MEFs are impaired in cell proliferation and Anp32b / mice was accompanied by the reduced size of oncogenic transformation. As seen in mixed-bred organs such as the heart, liver, spleen and kidney (Figures 1b Figure 1 Anp32b deficiency impairs normal cell proliferation and oncogenic transformation. (a) The body weight of 22 Anp32b +/+,30Anp32b +/ − and 8 Anp32b − / − mice at 4 weeks of age. Data were analyzed using Mann–Whitney U-test. **Po0.01. (b) Photographs of appearance and organs of Anp32b− / − mice and Anp32b+/+ littermate at 4 months of age. (c) The weight of organs from Anp32b+/+ and Anp32b− / − mice at 4 months of age. Data are presented as mean ± S.D. and significance is *Po0.05 (n = 4). (d) Western blots for the indicated protein in primary Anp32b+/+ and Anp32b− / − MEF cells. (e) Proliferation of primary Anp32b+/+ and Anp32b− / − MEFs was monitored for the indicated times. Nt/N0 represents the cell number at a given time normalized to the cell number at day 0. Data are presented as mean ± S.D. The experiment is representative of three separate experiments. (f) Western blots for the indicated protein in immortalized Anp32b+/+ and Anp32b− / − MEF cells. (g) Proliferation of immortalized Anp32b+/+ and − − Anp32b / MEFs was monitored for the indicated times. Data are presented as mean ± S.D. (h) Immortalized MEF cells were subcutaneously injected into nude mice and the size of masses were analyzed. Data are presented as mean ± S.D. Cell Death and Disease ANP32B deficiency suppresses proliferation and tumorigenesis S Yang et al 3 and c). To determine whether this is due to reduced cell ANP32B promotes cell cycle progression. To examine volumes or hypoplasia, histological H&E staining and flow whether the effect of ANP32B on breast cancer cell cytometric evaluation were performed. The results showed proliferation is partly due to cell cycle arrest, we used a that there was no obvious cell size difference between these double thymidine block to synchronize cells at the G1/S organs in Anp32b− / − and Anp32b+/+ mice (Supplementary border, followed by addition of nocodazole to block cells in Figure S1A and B). However, cell numbers in organs such G2/M. Flow cytometry analysis was then used to monitor the as spleen and thymus were dramatically decreased in progression of cells from G1/S to G2/M. The results showed Anp32b− / − mice (Supplementary Figure S1C), supporting that after nocodazole treatment within 3 h 89.5% of cells in that Anp32b deficiency causes a hypoplastic phenotype in shNC BT549 cells entered the S phase, whereas cells at the multiple organs. S phase only had 59.9 and 63.8%, respectively, in sh32b#1 To functionally characterize the role of Anp32b in normal and sh32b#2-infected cells. After nocodazole treatment for +/+ cell proliferation, we isolated MEFs from Anp32b and 9 h, only 46.3 and 39.8% of total cells entered the G2/M − − Anp32b / mice. The ANP32B protein was totally knocked phase in two sh32b-infected BT549 cells compared with out in Anp32b− / − MEF cells (Figure 1d). Cell proliferation 69.6% of total cells in shNC BT549 clones (Figure 3a). In assay showed that primary Anp32b− / − MEF cells
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